In recent years, in many olive oil producing countries, the research has been focused on the innovation of orchard management with the aims to reduce production costs and to increase yield and oil quality. For these purposes, attempts have been made to introduce intensive or superintensive orchards, or adopt new training systems (Connors et al., 2014; Freixa et al., 2011). In several countries, there has been a rapid increase of high-density olive cultivation regarding a limited number of mainly traditional cultivars, whereas possible new varieties with suitable characteristics are still under trial in some research institutions (Rallo et al., 2008; Rugini et al., 2016; Vivaldi et al., 2015). Moreover, the cultivars used, such as Arbequina, Koroneiki, and Arbosana, are not considered entirely acceptable by some countries, because the growers prefer to cultivate local varieties yielding high oil quality, although very few of them are suitable for intensive or superintensive plantations, mainly due to the excessive vigor of the plants. The establishment of suitable new genotypes by means of traditional crossbreeding requires a great deal of time. On the contrary, the planting of vigorous local cultivars, grafted on dwarfing rootstocks, could be a feasible strategy to achieve rapid results. However, the availability of dwarfing rootstocks is very limited and their effectiveness is genotype dependent (Fontanazza et al., 1998). It might be possible to select rootstocks from offspring of programmed crosses or in alternative to use varieties, since both do not require waiting a long time to reach the phenotypic stability of the adult phase as the seedling. Until now, little was known about the mechanisms involved in the dwarfing ability of rootstocks. Several hypotheses about the role of physiological stimuli and/or anatomical characters have been made (scion/rootstock disaffinity, water influence, hormonal factors, competition for carbohydrates or nutrients), each supported by experimental data. It was found that no single mechanism influences plant physiology. Rather, the vigor reduction induced by rootstock appears to be the result of a more complex interaction of various factors (Atkinson et al., 2003; Basile et al., 2003; Cohen and Naor, 2002; Gascó et al., 2007; Nardini et al., 2006; Solari et al., 2006; Soumelidou et al., 1994; Trifilò et al., 2007).
Mutagenesis induction is a useful technique for accelerating the genetic improvement of both varieties and rootstocks, but the difficulties observed in isolating stable mutants for vegetative propagation represent a strong deterrent. The first mutants in olive, showing different vegetative habits, were obtained ≈40 years ago, following the gamma ray irradiation of plants from Ascolana Tenera and Moraiolo cultivars. These mutants showed low agronomic value (Donini and Roselli, 1972) and cytogenetic instability. Subsequently, some mutants with compact phenotype from cultivars Leccino and Frantoio irradiated cuttings were able to reduce plant size when used as rootstock (Pannelli et al., 1990, 1992). Cytogenetic analyses showed that the mutants, named Frantoio Compact (FC) and Leccino Compact (LC), were mixoploids, whereas a mutant showing a dwarf vegetative habit, obtained from cv. Leccino and therefore named LD, proved to be the only stable diploid mutant (Rugini et al., 1996). The LD mutant produced fruits similar to those of the wild type genotypes, but bloomed at least 1 week later, while both FC and LC produced normal and large-sized fruits, suggesting that they originated from diploid or tetraploid cells, respectively, as validated by Rugini et al. (1996). Recently, Caporali et al. (2014) observed that most of the flowers produced by the mixoploid LC plants are tetraploids, characterized by floral structures larger than in the corresponding diploid plants, due to the increase of the cell size, which also occurred in the fruits. However, polyploidy had little effect on the LC fruit size, because these showed a less elongated shape than in cv. Leccino.
Diploid and tetraploid olive plants were isolated from the mixoploid LC and FC mutants by means of the in vitro shoot-tip fragmentation technique (Rugini et al., 1996). The tetraploid shoots were easily distinguished from the diploid or mixoploid ones, both in in vitro culture and in the greenhouse, at the early growing stages, due to their wider and thicker leaves.
It is well known that there are no tetraploid genotypes in the cultivated olive (O. europaea subsp. europaea); in fact, this is diploid with basic chromosome number n = 23, and a nuclear DNA content ranging from 2.3 pg/1C DNA in cultivars Leccino and Frantoio (Rugini et al., 1996), to 3.90 pg/2C in cv. Dolce Agogia and 4.66 pg/2C in cv. Pendolino (Bitonti et al., 1999). Loureiro et al. (2007) observed a nuclear DNA content ranging from 2.90 to 3.20 pg/2C in wild olive.
On the contrary, tetraploid and hexaploid individuals were detected respectively in the subspecies cerasiformis and maroccana of the olive complex O. europaea (Besnard et al., 2008; Rallo et al., 2003). In particular, it was hypothesized that tetraploid cerasiformis could be derived from the hybridization between the ancestors of the subspecies guanchica and europaea (Besnard et al., 2008; Rugini et al., 2011). Polyploidy is a very common condition in the plant kingdom, changing the organization and function of the genome at both genetic and epigenetic level (Comai, 2005). It is well known that polyploidization usually increases the cell dimensions, the fruit size, and sometimes the plants show a better adaptability than their diploid parents.
This study focuses on the vegetative and reproductive characterization of the stable tetraploid and diploid mutants obtained from the shoot apex fragmentation of mixoploid genotypes, grown under field conditions, and on the evaluation of their effect on the vigorous cultivar Canino when used as rootstocks.
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Reg (CEE) n. 2568/91 and Decreto MIPAAF 18/06/2014
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